A binding-triggered hybridization chain reaction cascade multi-site activated CRISPR/Cas12a signal amplification strategy for sensitive detection of α-synuclein.

Alpha-synuclein (α-syn) is closely related to the pathological process of Parkinson's disease (PD). Sensitive detection of α-syn is important for the early diagnosis and disease progression monitoring of PD. Herein, we report a binding-triggered hybridization chain reaction (HCR) cascade multi-site activated CRISPR/Cas12a signal amplification strategy for sensitive detection of α-syn. In this method, antibody-DNA capture probes recognized α-syn and bound with it to increase the local effective concentrations of two DNA strands, promoting their hybridization to form a split HCR trigger. Then the trigger initiated an HCR to generate a long double-stranded structure which contained abundant periodically repeated Cas12a/crRNA target sequences. Finally, the Cas12a/crRNA recognized the target sequence in HCR products and then the cleavage activity toward fluorescent reporters was activated, leading to the recovery of appreciable fluorescence signals. Our method provided a detection limit as low as 9.33 pM and exhibited satisfactory applicability in human serum samples. In summary, this study provides a homogeneous strategy for convenient, sensitive, and accurate detection of α-syn, showing great potential in the early diagnosis of PD.

An acid-responsive DNA hydrogel-mediated cascaded enzymatic nucleic acid amplification system for the sensitive imaging of alkaline phosphatase in living cells.

Alkaline phosphatase (ALP) is a class of hydrolase that catalyzes the dephosphorylation of phosphorylated species in biological tissues, playing an important role in many physiological and pathological processes. Sensitive imaging of ALP activity in living cells is contributory to the research on these processes. Herein, we propose an acid-responsive DNA hydrogel to deliver a cascaded enzymatic nucleic acid amplification system into cells for the sensitive imaging of intracellular ALP activity. The DNA hydrogel is formed by two kinds of Y-shaped DNA monomers and acid-responsive cytosine-rich linkers. The amplification system contained Bst DNA polymerase (Bst DP), Nt.BbvCI endonuclease, a Recognition Probe (RP, containing a DNAzyme sequence, a Nt.BbvCI recognition sequence, and a phosphate group at the 3'-end), and a Signal Probe (SP, containing a cleavage site for DNAzyme, Cy3 and BHQ2 at the two ends). The amplification system was trapped into the DNA hydrogel and taken up by cells, and the cytosine-rich linkers folded into a quadruplex i-motif in the acidic lysosomes, leading to the collapse of the hydrogel and releasing the amplification system. The phosphate groups on RPs were recognized and removed by the target ALP, triggering a polymerization-nicking cycle to produce large numbers of DNAzyme sequences, which then cleaved multiple SPs, restoring Cy3 fluorescence to indicate the ALP activity. This strategy achieved sensitive imaging of ALP in living HeLa, MCF-7, and NCM460 cells, and realized the sensitive detection of ALP in vitro with a detection limit of 2.0 × 10-5 U mL-1, providing a potential tool for the research of ALP-related physiological and pathological processes.

miR-21 inhibition reverses doxorubicin-resistance and inhibits PC3 human prostate cancer cells proliferation.

Many approaches have been examined to reversing multidrug resistance (MDR), but sub-optimal target-based strategies have limited their efficacy. Herein, we investigate microRNA (miR-21) suppression on the doxorubicin (DOX)-sensitisation of the DOX-resistant (PC3/DOX) cell line in prostate cancer (PCa). Expression levels of miR-21, P-glycoprotein (P-gp), MDR-1 and PTEN evaluated in PC3/DOX cancer cells by qRT-PCR and western blot analyses. The cytotoxic effects of transfected of miR-21 were assessed by MTT assay for 72 hr. Rhodamine123 (Rh123) assay was employed to define the activity of P-gp. Apoptosis was detected by Flow cytometry. As expected, miR-21 was expressed highly in PC3/DOX cells (p < 0.05). It was shown that miRNA-21 suppression considerably hindered PC3/DOX cell viability. miR-21 suppression dramatically downregulated P-gp expression and activity in DOX-resistance cells and abolished MDR by an increment of intracellular accumulation of DOX in PC3/DOX cells (p < 0.05). PTEN is a key modulator of the PI3K/Akt/P-gp cascade, which miR-21 suppression led to the upregulation of PTEN and sequentially lower-expression of P-gp that reversed MDR. Also, miR-21 repression enhanced the apoptosis rate of PC3/DOX cells. The findings of this paper contribute to the current understanding of the functions of miR-21 in MDR-reversing in PCa.

Hesperidin induces anticancer effects on human prostate cancer cells via ROS-mediated necrosis like cell death.

PURPOSE: Hesperidin, a plant-based molecule, has been shown to exhibit anticancer effects against the human prostate cancer cells. However, its mechanism of action is still unclear. This study was undertaken to investigate the mechanism underlying the anticancer effects of hesperidin against prostate cancer cells. METHODS: The CCK-8 kit-based proliferation analysis was performed to find out the effect of hesperidin administration on prostate cancer cell growth, in vitro. Apoptosis of cancer cells was studied with dual Annexin V-FITC/propidium iodide (PI) staining combined with flow cytometry. The latter was also used for the analysis of cancer cell mitotic cell cycle. The intracellular levels of reactive oxygen species (ROS) were determined with ROS-detection kit. Fluorescent probing was used for determination of mitochondrial membrane potential (MMP) of prostate cancer cells. The migration and invasion of cancer cells was studied using transwell assay. RESULTS: The in vitro treatment of prostate cancer cells led to significant decrease of cell growth and viability in a dose-dependent manner. The decline in the growth of cancer cells was shown to be resulting from initiation of cell cycle arrest and necrosis-like apoptotic cell death. The latter was shown to be triggered by intracellular accumulation of ROS molecules and reduction of MMP. Moreover, the hesperidin administration significantly reduced the cancer cell migration and invasion. CONCLUSION: Hesperidin was shown to selectively inhibit the growth of prostate cancer cells through apoptosis triggered by ROS generation. The results support its potential to act as lead molecule in anticancer drug design.

Risk factors for brain metastasis in patients with small cell lung cancer without prophylactic cranial irradiation.

BACKGROUND: This study aimed to determine the risk factors for brain metastasis (BM) and the prognostic factors for overall survival (OS) in patients with small cell lung cancer without prophylactic cranial irradiation (PCI). PATIENTS AND METHODS: Limited stage small cell lung cancer (LS-SCLC) patients achieving a complete response (CR) or partial response (PR) were enrolled into this study between January 2010 and December 2016. We retrospectively evaluated the influencing factors for time to BM and overall survival (OS). RESULTS: A total of 153 patients were enrolled into this study. Sixty-eight developed BM during the follow-up period. For the whole cohort, the 1­ and 2­year BM rates were 29.4 and 41.2%, respectively. Multivariate analysis showed that T stage (hazard ratio [HR] = 2.27, P = 0.024), neutrophil-to-lymphocyte ratio (NLR; HR = 2.07, P = 0.029), time to thoracic radiotherapy (HR = 0.34, P = 0.002) and chemotherapy cycles (HR = 0.49, P = 0.036) were the independent influencing factors of time to BM. Only NLR (HR = 2.11, P = 0.005) and time to thoracic radiotherapy (HR = 1.95, P = 0.011) were independent prognostic factors of OS. Of the 68 patients developing BM, those with BM occurring as the first relapse (42/68) had better OS than the others (39.5 months vs 23.0 months, P = 0.016). CONCLUSION: LS-SCLC patients without PCI had a high risk of BM. High T stage, high NLR, early thoracic radiotherapy and fewer chemotherapy cycles were the risk factors of BM. Further research is needed to confirm the results.

Role and mechanism of radiological protection cream in treating radiation dermatitis in rats.

OBJECTIVE: To explore the role and mechanism of a radiation protection cream (Rp) in the treatment of radiation dermatitis, and to accumulate necessary technical information for a new drug report on Rp. METHODS: High-performance liquid chromatography was used to establish the method of measuring the main effective ingredients of sovereign and adjuvant herbs of Rp drugs, and to formulate the draft quality standards of Rp. A total of 48 Sprague-Dawley male rats were randomly divided into the Model, Trolamine cream (Tc), Rp and Blank groups according to a random number table method. The skin of each rat's buttocks was irradiated using an electron linear accelerator to establish an acute radiation dermatitis model. The histological changes were observed under light microscopy and electron microscopy during wound healing and the effect of Rp on rat fibroblast Ku70/80 gene expression was detected at the transcriptional level. RESULTS: Pathological examination revealed that Rp protected the cellular and subcellular structures of skin after irradiation, promoting the proliferation and restoration of collagen fibers. Ku70/80 mRNA expression levels in the Rp and Tc groups were higher than that in the model group (P < 0.05). Moreover, The majority of grade radiation dermatitis relative to the Model, Rp and Tc groups for reducing grade III and IV dermatitis efficiency were 85.7% and 69.2% (P < 0.05), respectively. The efficacy of Rp group in treating radiation dermatitis was better than the Trolamine cream group by 16.5% (P < 0.05). CONCLUSION: Compared with Tc, Rp had certain advantages in the efficacy and performance to price ratio. Thus, Rp is considered an effective alternative formulation for the prevention and treatment of radiation dermatitis.

Rhizoma Pinelliae trypsin inhibitor separation, purification and inhibitory activity on the proliferation of BGC-823 gastric adenocarcinoma cells.

The aim of this study was to isolate and purify Rhizoma Pinelliae trypsin inhibitor (RPTI), determine its N-terminal amino acid sequence and evaluate its inhibitory effect on the proliferation of poorly differentiated BGC-823 human gastric adenocarcinoma cells. RPTI was separated and purified from a 40% (NH4)2SO4 precipitate of crude protein extract of Pinellia ternata tuber using affinity chromatography with trypsin as the ligand. The N-terminal amino acid sequence of RPTI was determined using the Edman degradation method. The inhibitory effect of RPTI on BGC-823 cell proliferation was detected in vitro using the MTT method and in vivo in tumour-bearing mice. The purified RPTI showed a single band under SDS-PAGE, its molecular weight was 14 kDa and its N-terminal amino acid sequence was DPVVDG. RPTI inhibited trypsin activity, with an inhibition ratio of 1:6.78 (mass). RPTI significantly inhibited the proliferation of BGC-823 cells in vitro. The IC50 of RPTI was 16.96 µg/ml within 48 h after treatment and 9.61 µg/ml within 72 h after treatment. Subcutaneous injection of RPTI around the tumour significantly inhibited BGC-823 tumour growth in mice. The tumour inhibitory effect was concentration- and dose-dependent. RPTI did not significantly influence the spleen coefficient of the mice. In conclusion, RPTI is a serine proteinase inhibitor with antitumour activity.